Although mechanical ventilation (MV) is a life-saving intervention in patients suffering from respiratory failure, prolonged MV is often associated with numerous complications including problematic weaning which increases patient morbidity and mortality. Prolonged MV results in a severely diminished blood flow and O2 delivery to the diaphragm at rest and during contractions, which limits O2 uptake and cripples contractile function. The latter would force reliance on anaerobic metabolism and lead to premature diaphragm fatigue and weaning failure. The mechanisms for the progressive reduction in diaphragm blood flow and the impaired hyperemic response during contractions with MV are unknown, but likely result from impaired microcirculatory function. To date, there are no studies investigating how prolonged MV affects respiratory muscle microcirculatory function in healthy individuals, let alone in aging or in patients with underlying pathologies. Our central hypothesis is that mechanical ventilation results in vascular dysfunction within the diaphragm and compromises the ability to augment O2 delivery and support contractile function to the diaphragm during weaning. Two integrated yet independent specific aims are proposed to test this central hypothesis. In Specific Aim 1, we will determine whether prolonged MV a) alters a vascular mechanical force in the diaphragm, which affects both endothelium responses and mechanical alterations (i.e., shear-stress), and b) impairs endothelial and/or smooth muscle function of the diaphragm resistance vasculature with MV and whether this is related to diminished NO signaling or oxidant production. For Specific Aim #2, we will employ two interventions designed specifically to target the unique environment of the diaphragm with MV and determine, in vivo, whether such interventions can preserve the blood flow and force generating capacity of the diaphragm after prolonged MV to facilitate weaning. We will use a combination of in vivo and in vitro approaches in these studies that will allow students to engage in semester long to multi-year projects in the laboratory. We predict that several of these projects will also provide the foundation for graduate and post-graduate studies of the students involved. This proposal is novel because it challenges the widespread assumption that diaphragm O2 delivery during MV is sufficient, and weaning difficulties are not due to vascular dysfunction. Information derived from this project could be rapidly translated to MV operational care to reduce the morbidity, mortality and health-care burden associated with MV and weaning difficulties.